The present invention relates to the field of green microalgae and their use in biotechnology. More particularly, the present invention describes the use of Chlamydomonas lacking a functional DYRKP-1 protein, for producing large amounts of neutral lipids (triacylglycerides: TAGs, or oils) and/or large amounts of starch, under stress conditions.
Because of their high biomass productivity and their ability to accumulate high intracellular amounts of starch (convertible into bioethanol), or oil (convertible into biodiesel), microalgae represent a promising feedstock for the production of next-generation biofuels (Hu et al., 2008; Wijffels and Barbosa, 2010). However, their productivity needs to be increased in order to reach sustainable biofuel production (Delrue et al., 2013).
Microalgae and, more generally, photosynthetic organisms have developed sophisticated strategies to optimize growth and survival under constantly fluctuating conditions of light, temperature and nutrient availability. In microalgae, deprivation of essential macronutrients strongly affects growth and induces drastic changes in the cellular metabolism. A general response to nitrogen or sulfur deprivation consists in a decrease in protein synthesis, an arrest in cell division, a massive accumulation of energy-rich storage compounds such as starch and triacylglycerols (Ball et al., 1990; Merchant et al., 2012), and a down-regulation of photosynthesis (Grossman, 2000; Peltier and Schmidt, 1991). This requirement of nutrient deprivation to trigger accumulation of reserve compounds is one of the major biological limitations of microalgae for biotechnology purposes because it impairs biomass productivity (Hu et al., 2008). Despite considerable interest for microalgae as a new feedstock (Larkum et al., 2012), little is known about signaling and regulatory genes and pathways controlling processes of photosynthetic energy conversion and storage in relation to nutrient and energy status.
Deciphering regulatory mechanisms controlling growth, photosynthesis and reserve accumulation in response to the nutrient and energy status is hence a key issue towards optimizing microalgal productivity for biotechnological applications.
With the aim to unravel regulatory mechanisms involved in the dynamics of reserve in response to nutrient availability, the inventors have now characterized one mutant of Chlamydomonas reinhardtii, screened on a defect in starch degradation and called std1 (for starch degradation). The std1 mutant harbors an insertion in a gene of the DYRK family, initially annotated as DYRK2 (Chlamydomonas genome version 4.0), and renamed here DYRKP-1. The Chlamydomonas std1 mutant, the first dyrk mutant of the green lineage reported so far, accumulates much more starch and oil than its wild-type progenitor in response to nutrient deprivation in photoautotrophic conditions, and more oil than its wild-type progenitor in response to nutrient deprivation also in mixotrophic conditions. The present invention hence provides methods to cultivate microalgae cells so as to optimize their growth for optimum production of starch and/or lipids from the microalgae. The methods of the present invention induce and enhance accumulation of starch and/or oil, depending on the culture conditions, within the microalgae cells. The methods disclosed are suitable for large-scale production of starch- and/or oil-rich microalgae.
A first aspect of the present invention is hence a method for producing biomass feedstock, comprising the steps of:
(i) cultivating green microalgae cells in which the expression and/or the activity of the DYRKP-1 protein is altered; and
(ii) inducing reserve accumulation and/or increase in biomass production by said microalgae.
As defined herein, the “DYRKP-1 protein” is a DYRK protein expressed by microalgae, which possesses a DH-box having the following sequence: H(R/K)TGFEEXK(D/E/N)(F/L) (SEQ ID No: 3). The amino acid sequence and coding sequence (cDNA sequence including the 5′- and 3′-UTRs) of the DYRKP-1 protein of Chlamydomonas reinhardtii are disclosed herein (SEQ ID NO: 1 and 2, respectively). From these sequences, the skilled artisan can perfectly identify the sequence of DYRKP-1 in any green microalga different from Chlamydomonas reinhardtii, by identifying in said microalga the protein homologous to that of SEQ ID NO: 1. In the present text, a protein is considered as being an homolog of DYRKP-1 from Chlamydomonas reinhardtii if both proteins share a common ancestor, as shown by very similar primary sequences (at least 50, 60, 70, 75, 80, 85, 90, 95 or 99% identity, as measured by BLAST) and secondary and tertiary structures.
In the present text, “altered” or “impaired” means that the expression and/or the activity of the DYRKP-1 protein is changed, so that the activity of the protein is decreased. For example, the DYRKP-1 gene can be silenced, knocked down, mutated and/or interrupted, so that the microalgae lack a functional DYRKP-1 protein. Activity of the DYRK-P protein could also be inhibited by chemical compounds acting as specific inhibitors.
As disclosed in the experimental part below, the method according to the invention can be performed with Chlamydomonas, especially with Chlamydomonas reinhardtii. 
In step (i), the cells are cultured according to any usual protocol known by the skilled artisan. For example, they can be grown photoautotrophically in a MOPS-buffered minimal medium (MM) supplied with 2% CO2 to a density of 2-5×106 cells/nil.
In a particular embodiment of this aspect, said inducing reserve accumulation comprises incubating the microalgae cells in a deficient medium, i.e., a medium which does not comprise, in sufficient quantities, all the nutrients required for optimal growth of green microalgae. Examples of such a deficient medium include a medium deficient in at least one element selected from the group consisting of nitrogen, sulfur and phosphorus, in a form which can be metabolized by the microalgae. Of course, the phrase “deficient in” is not to be read in an absolute sense (i.e., with a concentration equal to zero), but means that the concentration of said nutrient in the medium is far below (at least 10-fold below) the concentration of said nutrient in a classical medium used for microalgae culture.
Between step (i) and step (ii), the cells can be harvested and transferred into the deficient medium. Alternatively, typically in a continuous culture device, the deficit in the medium is created by the cell's metabolism, in absence of exogenous addition of at least one nutrient. For example, as illustrated in the experimental part and in FIG. 9A, addition of minimal N-free medium in a photobioreactor operated as a turbidostat (to maintain the cellular biomass at a constant level) resulted in a decrease in the ammonia content of the culture medium, which was fully exhausted in less than 2 days.
In a particular embodiment, the step of inducing reserve accumulation comprises illuminating the microalgae cells with a light enabling photosynthesis to occur.
For example, this illumination can be performed at an intensity of at least 25 μmol photons m−2 s−1, or at least 100 μmol photons m−2 s−1, for example comprised between 25 and 2000 μmol photons m−2 s−1, during 8 to 24 hours per day. The skilled artisan perfectly knows that the effects of the light intensity depend in fact on the intensity which is really received by the microalgae, and hence depend on several parameters such as cell density and the shape of the photobioreactor, etc., and is able to adapt the illumination intensity to the specific encountered conditions.
Remarkably, the inventors have shown that nutrient deprivation does not lead to a rapid stop of photosynthesis by microalgae lacking DYRKP-1 activity, as is usually the case for wild-type microalgae. This is particularly advantageous, since the cells can not only be oil- and/or starch-enriched, but in addition, the global biomass increases during several days of deficient conditions, leading to remarkable overall increased lipid and starch productivity.
Hence, in an advantageous embodiment of the present invention, the step of incubating the microalgae cells in a deficient medium lasts at least 24 hours, for example from 2 to 8 days, preferably from 3 to 6 days. Of course, the cell growth in a deficient medium strongly depends on experimental conditions, particularly of the cell density, and hence the skilled artisan will adapt the duration of the incubation with a deficient medium so that, under the specific conditions used, the reserve accumulation and/or biomass increase is optimal.
According to a particular embodiment of the invention, step (ii) comprises incubating the microalgae cells in a medium comprising organic carbon such as, for example, acetate. According to a non-limitative example of such mixotrophic conditions, illustrated in the examples below, the cells are incubated in step (ii) during 2 to 6 days in a nitrogen-deficient medium comprising acetate, under constant illumination of at least 50 μmol photons m−2 s−1. The inventors have shown that in mixotrophic conditions, microalgae lacking DYRKP-1 activity respond to nutrient depletion by increased lipid accumulation compared to wild-type cells. Hence, this particular embodiment of the method is advantageously used for producing oil, for example for biodiesel production.
According to another embodiment, the inducing step (ii) comprises incubating the microalgae cells in a deficient medium as defined previously and in photoautotrophic conditions, i.e., in conditions such that they convert radiant energy into biologically useful energy and synthesize metabolic compounds using only carbon dioxide, bicarbonate or carbonates as source of carbon. Typically, the microalgae cells are incubated under illumination in a medium essentially devoid of organic carbon which they can metabolize. In what precedes, “essentially devoid of organic carbon” means that no organic carbon which can be metabolized by the microalgae has been added into the medium. According to a preferred version of this embodiment, the cells are incubated in step (ii) during at least 15 hours, preferably at least 1, 2 or 3 days, and up to 6 or more days in a nutrient-deficient medium, for example in a nitrogen-deficient medium essentially devoid of organic carbon which can be metabolized by the microalgae. The inventors have shown that in photoautotrophic conditions, microalgae lacking DYRKP-1 activity accumulate much more starch and oil than their wild-type progenitor in response to nutrient deprivation. Hence, this particular embodiment of the method is advantageously used for producing starch, for example for bioethanol production, as well as for producing oil, for example for biodiesel production. This embodiment is particularly interesting, because in photoautotrophic conditions, cells can supply their need for carbon completely through photosynthesis, which is a major advantage compared to cells requiring an additionally supplied carbon source for growth (such as yeast or E coli).
It is to be noted that microalgae naturally produce polyunsaturated fats (omega-3 and omega-6), as well as complex molecules such as carotenoids, and that these high-value products can also be produced according to the methods described herein. The invention hence also relates to methods for producing fatty acids, polyunsaturated fats, carotenoids and other compounds for cosmetic and/or pharmaceutical industries, as well as food supplements, comprising a step of starch and/or triacylglycerols accumulation in microalgae by performing a method as described above
The methods of the invention may comprise one or more extraction steps after the triggering of starch and/or triacylglycerols accumulation step in microalgae. The extraction step may be implemented using solvents or another extraction method well known form the skilled artisan.
The present invention will be understood more clearly from the further description which follows, which refers to examples illustrating the response of microalgae lacking DYRKP-1 activity to nutrient deprivation, as well as to the appended figures.